The present inventions involves the large-scale production of membrane specific proteins and more particularly involves the production of such proteins using transgenic animals and most specifically involves the production of CFTR, the protein involved with Cystic Fibrosis.
Striking progress has been made in our understanding of cystic fibrosis (CF) during the period since the gene associated with the disease was identified1-3. Many mutations within the gene have been identified in DNA from patients with CF4-10. The protein product of the gene, named CFTR, has been identified11 and functional studies have shown that CFTR cDNA is able to complement the defect in ion transport characteristic of cells from CF patients12,13. The domain structure of CFTR has been probed by analysis of mutated versions of the protein14-18. Such studies indicate that CFTR is a Clxe2x88x92 channel14,19,20 and that it is required by phosphorylation of the R-domain16,18,21 and by nucleotide binding22. Other studies suggest that the molecular basis of the defect in mutant forms of CFTR associated with the most common CF genotypes is the inability of the nascent protein to traffic within the cell15,17. While these results are fascinating, further understanding of the cystic fibrosis disease condition and progress toward therapy, in particular, protein replacement therapy has been severely hampered by the unavailability of the CFTR protein. Although the protein has been produced in a number of different recombinant systems11,12,15,19-21, the level of expression has been quite modest, perhaps because there is an upper limit on the amount of a functional Cxe2x88x92 channel that can be produced in a given cell. It is one aspect of the present invention to is develop an alternative means of CFTR expression.
It is yet another aspect of the present invention to employ plasma membrane vesicles that are shed from the host cell.
It is yet another aspect of the present invention to sequester nascent CFTR into such plasma membrane vesicles. Apocrine like secretion of endogenous proteins has been reported in the synthesis of milk fat globules by cells of the mammary epithelia23-25, however to our knowledge none of such proteins have involved exogenous or recombinant membrane proteins.
In accordance with the principles of the present invention it has been surprisingly discovered that exogenous or recombinant membrane proteins can be produced in the milk of transgenic animals. In the most preferred embodiment of the present invention, recombinant CFTR has been produced in the milk of transgenic mice containing CFTR cDNA downstream of a mammary specific promoter. Most preferred embodiments will employ larger transgenic animals including, for example, rabbits, goats, sheep and cows which produce large quantities of milk however, the development of such animals takes considerably longer than the time periods required for developing transgenic mice. While the time periods vary considerably, the procedures and methods are substantially identical.
Other embodiments include the apocrine like secretion in tissue culture of membrane proteins by cells which have been maintained in a differentiated state. Such proteins would be secreted as part of the membrane released during the xe2x80x9cpinching offxe2x80x9d process. Membrane proteins suitable for production according to the methods of the present invention include receptors, channels, viral glycoproteins, transporters and other proteins typically associated with cellular membranes. Such membrane proteins can be used for therapeutics such as by administration of the abnormally deficient or missing protein, or by use as vaccines in the case of viral glycoproteins including for example utilization of the envelope of HIV, Herpes Virus or influenza in order to develop immunity with respect to infection caused thereby.
Advantageously, methods of the present invention can also be employed for the production of membrane proteins useful for diagnostic purposes. For example, one could use the present methods to produce the receptor for thyroid stimulating hormone (TSHR) which would be useful for the diagnosis of Graves Disease. Such proteins could also be used to screen for therapeutically active compounds.
Preferred methods of the present invention will employ promoters, ideally coupled with suitable enhancers, that are mammary specific so that production of the desired membrane protein is incorporated into milk fat globules which will appear in the milk of a lactating female transgenic animal. Such an animal is, of course, by definition, reproductively competent. Greater appreciation of these and other embodiments will be acquired by study of the following drawings, examples and detailed procedures.